U.S. patent number 11,154,022 [Application Number 16/201,262] was granted by the patent office on 2021-10-26 for production of haploid lolium.
This patent grant is currently assigned to The United States of America, as represented by the Secretary of Agriculture. The grantee listed for this patent is The United States of America, as represented by the Secretary of Agriculture, The United States of America, as represented by the Secretary of Agriculture. Invention is credited to Bryan K. Kindiger.
United States Patent |
11,154,022 |
Kindiger |
October 26, 2021 |
Production of haploid lolium
Abstract
A method for producing haploid Lolium plants may start with
providing a Lolium multiflorum inducer line, the L. multiflorum
inducer line having the ability to induce mitotic genome
instability and haploid sectoring when hybridized as a maternal
parent with a Lolium sp. paternal parent, such as previously
disclosed lines IL1 and IL2. The inducer line may then be crossed
with a Lolium sp. to generate F1 plants, and the F1 plants may be
self-fertilized so as to recover seed from the selfed plant. The
recovered seed may then be planted to generate one or more F2
plants, and at least one of the F2 plants may be a haploid Lolium
plant.
Inventors: |
Kindiger; Bryan K. (El Reno,
OK) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture |
Washington |
DC |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary of Agriculture (Washington,
DC)
|
Family
ID: |
1000005890444 |
Appl.
No.: |
16/201,262 |
Filed: |
November 27, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200163299 A1 |
May 28, 2020 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01H
5/12 (20130101); A01H 1/08 (20130101); A01H
6/463 (20180501) |
Current International
Class: |
A01H
1/08 (20060101); A01H 6/46 (20180101); A01H
5/12 (20180101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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102224801 |
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Oct 2011 |
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CN |
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103081797 |
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May 2013 |
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CN |
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106171965 |
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Aug 2018 |
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CN |
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Other References
Kindiger (Jan. 2012, "Notification of the Release of Annual
Ryegrass Genetic Stock IL1", Journal of Plant Registrations 6(1):
217-220). cited by examiner .
Kindiger (Jan. 2012, "Notification of the Release of Annual
Ryegrass Genetic Stock IL1", Journal of Plant Registrations 6(1):
217-220). (Year: 2012). cited by examiner .
International Search Report for PCT/US2019/062566 dated Mar. 13,
2020. cited by applicant .
Written Opinion for PCT/US2019/062566 dated Mar. 13, 2020. cited by
applicant.
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Primary Examiner: Kallis; Russell
Attorney, Agent or Firm: Fado; John Atkinson; Ariel
Claims
What is claimed is:
1. A method for producing haploid Lolium plants, the method
comprising: providing a Lolium mutiflorurn inducer line, the L.
multiflorum inducer line having the ability to induce mitotic
genome instability and haploid sectoring when hybridized as a
maternal parent with a Lolium sp. paternal parent; crossing said L.
multiflorum inducer line with a Lolium sp. to generate F1 plants;
self-fertilizing at least one of the generated F1 plants;
recovering and planting seed from the at least one self-fertilized
F1 plant to generate one or more F2 plants; and growing said one or
more F2 plants, wherein at least one of the one or more F2 plants
is a haploid Lolium plant.
2. The method of claim 1, wherein the L. multiflorum inducer line
is one of: IL1 (ATCC deposit accession no. PTA-10229), IL2 (ATCC
deposit accession no. PTA-10315), and progeny thereof.
3. The method of claim 1, wherein in crossing the L. multiflorum
inducer line with a Lolium sp. to generate F1 plants, the L.
multiflorum inducer line is the maternal parent.
4. The method of claim 1, wherein the Lolium sp. is one of L.
multiflorum and L. perenne.
5. The method of claim 1, further comprising recovering at least
one of the one or more F2 plants.
Description
BACKGROUND
Haploid induction systems result in the generation of either
maternal or paternal haploids that can efficiently advance genetic
gains through gamete selection or a dihaploid production breeding
or selection program. The standard approach for the production of
haploids in Lolium sp. is almost exclusively limited to microspore
or anther culture approaches (Palmer and Keller, Challenges and
Limitations to the use of Haploidy in Crop Improvement,
Biotechnology in Agriculture and Forestry, Vol. 56, pp. 295-303,
2005; Tuvesso et al. Molecular markers and doubled haploids in
European plant breeding programmes, Euphytica 58:305-312, 2007;
Dunwell, Haploids in flowering plants: Origins and exploitation,
Biotech. J. 8:377-424, 2010). Haploidy, as generated through
microspore or anther culture, is highly labor intensive, expensive
and restricted by necessitating the plant being cultured to possess
a genotype amendable to such methodology. Within the Lolium genus,
microspore derived levels of haploid generation are known to range
from 1-5%. Such haploids, when identified, are then subjected to a
mitotic inhibitor in order to double the chromosome number to
produce homozygous or dihaploid (DH) lines. These materials can
then be utilized for advanced selection or breeding methodologies
or molecular marker assisted breeding techniques (Martinez et al.,
On the use of double haploids for detecting QTL in outbred
populations, Heredity 88:423-431, 2002; Chang and Coe, Doubled
haploids, Biotechnology in Agriculture and Forestry. Vol. 63, pp.
127-142, 2009; Begheyn et al. Review: Haploid and Double Haploid
Techniques in Perennial Ryegrass (Lolium perenne L.) to Advance
Research and Breeding, Agron. 60:1-17, 2016). Several recent
reviews regarding the generation and utilization of haploids and
dihaploids in Lolium sp. have been published and significant
details on the history, methods and outcomes of that prior research
can be gleaned from those reviews (Chang and Coe, 2009; Geiger,
Doubled haploids, Maize Handbook. Vol. II: Genetics and Genomics,
pp. 641-659, 2009; Dunwell, 2010; and Begheyn et al., 2016).
In other instances, inducer lines (Fehr, Homozygous lines from
double haploids, Principles of cultivar development. Vol. 1, pp:
337-358. 1984) can be utilized to produce haploids and eventually
DH lines possessing only the maternal genotype that can then be
eventually introduced into breeding programs (Forster and Thomas,
Doubled haploids in genetics and plant breeding, Plant Breed Rev.
25:57-88, 2005). In addition, there are systems where paternal
haploids are generated when the maternal or egg parent represents
the inducer line, such as seen with the indeterminate gametophyte
system of maize (Kermicle, Androgenesis Conditioned by a Mutation
in Maize, Science 166:1422-1424, 1969; Rotarenco and Chalyk,
Selection at the level of haploid sporophyte and its influence on
the traits of diploid plants in maize, Genetika 32:479-485, 2000).
Each of the above methods produces haploids through novel meiotic
behaviors found in the respective lines.
Previously, an approach utilizing annual ryegrass (Lolium perenne
L. subsp. multiflorum (Lam.) Husnot (syn. Lolium multiflorum Lam.)
lines called IL1 and IL2 (Kindiger and Singh, Registration of
Annual Ryegrass Genetic Stock IL2, J. of Plant Reg. 5:254-256,
2011; Kindiger, Notification of the Release of Annual Ryegrass
Genetic Stock IL1, J. of Plant Reg. 6:117-120, 2012) has been
described. IL1 and IL2 have the ability to induce mitotic genome
instability and haploid sectoring when hybridized with tall fescue
(Lolium arundinaceum (Schreb.) Darbysh.) (syn. Festuca arundinacea
Schreb.) (Kindiger, Generation of Paternal Dihaploids in Tall
fescue, Grassland Science. 62:243-247, 2016).
All of the references cited herein, including U.S. Patents and U.S.
Patent Application Publications, are incorporated by reference in
their entirety.
Mention of trade names or commercial products in this publication
is solely for the purpose of providing specific information and
does not imply recommendation or endorsement by the U.S. Department
of Agriculture.
SUMMARY
This research describes the generation of haploid genotypes
following the hybridization of L. multiflorum or L. perenne
genotypes to an inducer line, herein also referred to as an IL
line. The principal advantage to this haploid inducement approach
is that microspore methods or other culture approaches are not
utilized.
According to one embodiment of the invention, a method for
producing haploid Lolium plants may include: (1) providing a Lolium
multiflorum inducer line, the L. multiflorum inducer line having
the ability to induce mitotic genome instability and haploid
sectoring when hybridized as a maternal parent with a Lolium sp.
paternal parent, (2) crossing the L. multiflorum inducer line with
a Lolium sp. to generate F1 plants, (3) self-fertilizing at least
one of the generated F1 plants, (4) recovering and planting seed
from the at least one self-fertilized F1 plant to generate one or
more F2 plants, and (5) growing said one or more F2 plants, and at
least one of the one or more F2 plants is a haploid Lolium
plant.
According to a further aspect of the invention, the L. multiflorum
inducer line is one of: IL1 (ATCC deposit accession no. PTA-10229)
and IL2 (ATCC deposit accession no. PTA-10315).
According to a further aspect of the invention, the L. multiflorum
inducer line is used as the maternal parent.
Another embodiment of the invention may be the F2 haploid Lolium
plant produced by the above method.
BRIEF DESCRIPTION OF THE FIGURES
Advantages of embodiments of the present invention will be apparent
from the following detailed description of the exemplary
embodiments. The following detailed description should be
considered in conjunction with the accompanying FIGURES in
which:
Exemplary FIG. 1A shows a flow cytogram showing the presence of a
large haploid sector in an F1 hybrid, as well as a small sector
indicating a tetraploid recovery.
Exemplary FIG. 1B shows a flow cytogram of an individual with a
ryegrass phenotype exhibiting a purely haploid genome peak.
STATEMENT REGARDING DEPOSIT OF BIOLOGICAL MATERIAL UNDER THE TERMS
OF THE BUDAPEST TREATY
A sample of at least 2,500 seeds of each of the Lolium multiflorum
lines referred to herein as inducer line IL-1 and inducer line
IL-2, were deposited under the conditions of the Budapest Treaty
with the American Type Culture Collection (10801 University Blvd,
Manassas, Va., 20110-2209, USA) on Jul. 22, 2009, and Aug. 28,
2009, respectively, and were assigned deposit accession nos. ATCC
PTA-10229 and ATCC PTA-10315, respectively. The lines have no
restrictions on their availability to the public.
DETAILED DESCRIPTION
Aspects of the invention are disclosed in the following description
and related drawings directed to specific embodiments of the
invention. Alternate embodiments may be devised without departing
from the spirit or the scope of the invention. Additionally,
well-known elements of exemplary embodiments of the invention will
not be described in detail or will be omitted so as not to obscure
the relevant details of the invention. Further, to facilitate an
understanding of the description discussion of several terms used
herein follows.
As used herein, the word "exemplary" means "serving as an example,
instance or illustration." The embodiments described herein are not
limiting, but rather are exemplary only. It should be understood
that the described embodiments are not necessarily to be construed
as preferred or advantageous over other embodiments. Moreover, the
terms "embodiments of the invention", "embodiments" or "invention"
do not require that all embodiments of the invention include the
discussed feature, advantage or mode of operation.
"Optional" or "optionally" means that the subsequently described
event or circumstance may or may not occur, and that the
description includes instances in which said event or circumstance
occurs and instances where it does not. For example, the phrase
"optionally comprising X" means that the composition may or may not
contain X, and that this description includes compositions that
contain and do not contain X.
The term "consisting essentially of" excludes additional method (or
process) steps or composition components that substantially
interfere with the intended activity of the method (or process) or
composition, and can be readily determined by those skilled in the
art (for example, from a consideration of this specification or
practice of the invention disclosed herein).
The invention illustratively disclosed herein suitably may be
practiced in the absence of any element (e.g., method (or process)
steps or composition components) which is not specifically
disclosed herein.
According to at least one exemplary embodiment, a haploid Lolium
plant may be produced by first generating a hybrid between an
inducer line (IL) and either of a L. multiflorum or a L. perenne.
The resulting F1 hybrid may then be selfed, and the resulting seed
planted to create F2 plants, at least some of which may be haploid
Loliums.
The IL used may be any suitable inducer line, such as an inducer
line that has the ability to induce mitotic genome instability and
haploid sectoring when hybridized with the Lolium paternal parent,
for example the IL1 or IL2 lines as described in Kindiger and
Singh, 2011; Kindiger, 2012; U.S. Pat. No. 8,618,353; and
accessible via. ATCC deposit accession no. PTA-10229, and suitable
progeny thereof.
Examples
Generation of Haploids
Hybrids were generated in 2015 at the Barenbrug Research
Laboratory, Albany, Oreg. utilizing the IL lines as the maternal
parent. The ryegrass genotypes utilized represented a small but
diverse group of tetraploid L. perenne and L. multiflorum
genotypes. Hybridizations were made by crossing inducer line
IL1.times.LPT3A99 cv. (4n=4.times.=28); IL1.times.Jumbo cv.
(4n=4.times.=28); and IL1.times.Remington cv. (4n=4.times.=28)
(Table 1). LPT3A99 represents a perennial tetraploid experimental
ryegrass. Jumbo represents a tetraploid, annual ryegrass. The
Remington cultivar represents an intermediate, tetraploid perennial
form of ryegrass. All materials were provided by Barenbrug Seeds,
West Coast Research Laboratory, Albany, Oreg. USA.
Six of the generated IL.times.ryegrass F1 were submitted to
chromosome counts and then selfed. The generated F2 seed were sent
for analysis at the USDA-ARS, Grazinglands Research Laboratory, El
Reno, Okla. USA. The experimental cultivar LPT3A99 provided four
individuals for this study. One IL.times.LPT3A99 F1 possessed a
tetraploid constitution (4n=4.times.=28). Three additional
IL.times.LPT3A99 F1 exhibited a diploid (2n=2.times.=14) genome
constitution. The IL.times.Jumbo hybridization produced one
individual with a diploid (2n=2.times.=14) constitution. The
IL.times.Remington produced one F1 with a diploid (2n=2.times.-=14)
constitution.
At maturity, the F1 inflorescences were broken up by hand followed
by a light cleaning to remove stems. The resulting F2 seed were
then placed in trays containing a light potting soil mix for
germination and seedling development. Following two to three weeks
of germination, seedlings appeared and were allowed to grow to an
appropriate size that allowed the phenotypic diversity to be
observed. As the plants matured, the recovered seedlings were
submitted to both chromosome root-tip counts (Kindiger, A technique
for the preparation of Somatic Chromosomes of Maize, The Maize
Handbook, 74:481-483, 1996) and flow cytometry analysis. The
seedlings were eventually transferred to larger six-inch pots to
allow more growth and additional phenotypic observation ( ). Over
40 F2 seed were evaluated from each F1 individual (Table 1).
TABLE-US-00001 TABLE 1 Offspring produced from the various IL
.times. Lolium sp. Hybridizations Number and type of F2 F1 Hybrid
F1 Ploidy Haploid Diploid Triploid Tetraploid Mixoploid IL/LPT3A99
4n 1 43 3 2 2 IL/LPT3A99 2n 10 30 2 0 0 IL/LPT3A99 2n 5 43 0 0 1
IL/LPT3A99 2n 8 35 8 0 0 IL/Jumbo 2n 10 37 3 0 0 IL/Remington 2n 14
33 3 0 0 Totals: 48 269 19 2 3
Plant Material Analysis
Flow cytometry was used to analyze the ploidy of the plant hybrids
produced in this study, based on published methods (Galbraith et
al., Rapid flow cytometric analysis of the cell-cycle in intact
plant tissues, Science 220:1049-1051, 1983; Cousin et al., An
efficient high-throughput flow cytometric method for estimating DNA
ploidy level in plants, Cytometry 75:1015-1019, 2009). Flow
cytometry was performed on an Attune NxT flow cytometry under the
following conditions and protocols.
In 2017, mature leaf samples from the greenhouse grown materials
were obtained from each F1 and F2 individuals to determine if the
degree of any potential genome or somatic loss could be detected.
In most instances leaf samples identified obvious plant sectors
through flow cytometry analysis.
For the flow cytometric analysis, approximately 0.05 g of fresh cut
leaf tissue were placed in 1.5 ml Eppendorf tubes. Approximately
0.05 g of a 0.9-2.0 maceration stainless steel bead product
(SSB14B, Next Advance Inc., Averill Park, N.Y., USA) and one 3.2 mm
stainless steel bead (SSB32, Next Advance Inc., Averill Park, N.Y.,
USA) were combined for leaf maceration. 500 .mu.L of Galbraith
solution was placed in each tube (Galbraith et al., 1983) and each
sample was placed in a rotary bullet blender tissue homogenizer
(Next Advance Inc., Averill Park, N.Y., USA) using the
manufacturer's suggested settings to macerate the leaf tissue.
Approximately 400 .mu.L of this fluid were transferred from the
Eppendorf tubes to 15 ml Corning tubes. Nuclei labelling and
detection were achieved by dispensing 1 ml of the FxCycle PI/RNase
staining solution (Invitrogen by Thermo Fisher Scientific, 81 Wyman
Street, Waltham, Mass. 02451 USA) into the macerated leaf tissue
for one hour. Following the manufacturer's staining
recommendations, samples were retained in darkness during the
one-hour staining interval. Prior to flow cytometric analysis each
sample was filtered through a 50 .mu.m CellTrics disposable filter
(Sysmex-Partec GmbH, Goerlitz, Germany) before evaluations in a
flow cytometer (Model AFC2, Thermofisher Scientific, 81 Wyman
Street, Waltham, Mass. 02451 USA).
The flow cytometer was set to deliver a volume of 100 .mu.L from
the sample syringe at a flow rate of 25 .mu.L/min. The cytometer
software parameters were initiated at the "start" mode for a few
seconds, prior to initiating the "record" mode. This step was
performed to stabilize the sample rate and equilibrate the
concentration of the dye bound to the sample nuclei. The threshold
of the forward scattering (FSC) detector was placed in the "or"
mode while the side scattering and fluorescence detectors were
placed in the "ignore" mode. Default threshold settings were
utilized for the logic control box (OR) and the forward scattering
channel detector (FSC) was set at a threshold of 25.0.times.1000.
The number of recorded events was set to 1000 with the particular
event peaks being gated and average median fluorescent values were
used to estimate the ploidy/genome size of the nuclei being
recorded. To provide for a base line estimator for various genome
size estimations, the IL1 inducer line and the Lolium cultivars
LPT3A99, Jumbo, and Remington were used as controls.
The results of the flow cytometry analysis are shown above in Table
1. Exemplary flow cytograms are shown in FIGS. 1A and 1B. In the
images, the height of the peak is representative of the number of
nuclei scanned possessing any particular nuclear genome size. The
higher the peak, the greater the number of that particular class of
nuclei. The large peaks on the left side represent haploid genome
peaks. The smaller peak to the right in FIG. 1A illustrates the
tetraploid F1 hybrid IL1.times.LPT3A99.
The genome instability and the generation of pure haploid genotypes
were further confirmed by chromosome root-tip counts (data not
shown).
Haploidy in recoveries utilizing the inducer lines for a L. perenne
hybridization have been observed to range from 10% to 27.5% (Table
1). In total in the above examples, over 48 haploids were derived
from 341 F2 selfs. It was observed that there was a difference in
frequency of haploid generation across genotypes.
The foregoing description and accompanying FIGURES illustrate the
principles, preferred embodiments and modes of operation of the
invention. However, the invention should not be construed as being
limited to the particular embodiments discussed above. Additional
variations of the embodiments discussed above will be appreciated
by those skilled in the art.
Therefore, the above-described embodiments should be regarded as
illustrative rather than restrictive. Accordingly, it should be
appreciated that variations to those embodiments can be made by
those skilled in the art without departing from the scope of the
invention as defined by the following claims.
* * * * *